Lakkakula Satish

Influence of plant growth regulators and spermidine on somatic embryogenesis and plant regeneration in four Indian genotypes of finger millet (Eleusine coracana (L.) Gaertn)

Abstract Effect of plant growth regulators and spermidine on somatic embryogenesis and regeneration was investigated in finger millet. Mature embryos, and 3 days old seedling-derived shoot apical meristems were cultured on Murashige and Skoog (MS) medium containing picloram, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid. Improved embryogenesis (84.7xa0%) was found on MS medium containing 4.0xa0mgxa0L−1 2,4-D and 0.5xa0mgxa0L−1 kinetin in both explants. MS medium containing 1.5xa0mM spermidine along with 4.0xa0mgxa0L−1 2,4-D and 0.5xa0mgxa0L−1 kinetin produced highest frequency (90.4xa0%) of somatic embryogenesis from mature embryo derived callus in genotype ‘CO(Ra)-14’. On same medium somatic embryogenesis frequencies of ‘GPU-25’, ‘Try-1’ and ‘Piyur-2’ genotypes were 55.5, 85.3 and 58.7xa0%, respectively after 4xa0weeks of incubation in dark. MS medium containing 4.0xa0mgxa0L−1 6-benzylaminopurine, 0.2xa0mgxa0L−1 2,4-D and 1.5xa0mM spermidine was found to be optimum for shoot regeneration or somatic embryos in all four genotypes of finger millet. We also studied the influence of exogenous spermidine (2.0–4.0xa0mM) on regeneration of ‘CO(Ra)-14’ mature embryo-derived new and long-term (20–180 daysxa0old) calluses. Highest regeneration frequency 93.1xa0% and mean number of shoots 25.5 were produced on MS medium containing 3.0xa0mM spermidine, 4.0xa0mgxa0L−1 BAP and 0.2xa0mgxa0L−1 2,4-D using 60 days old callus. Regenerated shoots effectively rooted on half-strength MS medium and successfully acclimatized in soil with 100xa0% survival rate and they grew normally without showing any morphological variation. Genetic variation of in vitro derived plants and control plants were analyzed by RAPD markers.

Direct plant regeneration from in vitro-derived shoot apical meristems of finger millet (Eleusine coracana (L.) Gaertn.)

A rapid and reproducible protocol for direct plant regeneration has been established using in vitro-derived, actively growing shoot apical meristems (SAMs) of the genotype CO(Ra)-14. Shoot apical meristems from 3-day-old seedlings of three finger millet genotypes viz, CO(Ra)-14, Try-1, and Paiyur-2 were assessed for the efficiency of direct shoot induction. The regeneration efficiency of CO(Ra)-14 surpassed the other two genotypes and produced a mass of green, multiple shoots within 12xa0d on Murashige and Skoog medium containing 17.6xa0μM 6-benzylaminopurine (BA), 0.9xa0μM 2,4-dichlorophenoxyacetic acid in combination with 750xa0mgxa0L−1 proline, 500xa0mgxa0L−1 casein enzymatic hydrolysate, and 2xa0mgxa0L−1 glycine. CO(Ra)-14 showed a maximum frequency of multiple shoot regeneration (96%) with an average of 8.3 shoots per explant. The age and size of shoot apical meristems played a crucial role in triggering adventitious shoot bud proliferation. The multiple shoot buds elongated spontaneously upon reducing the concentration of BA (to 11.0xa0μM) in the shoot induction medium. The in vitro proliferated shoots were rooted best in half-strength MS medium containing 2.8xa0μM indole-3-acetic acid and successfully acclimated in the field, subsequently developing into fertile plants. Thus, we report a short tenure (45xa0d) in vitro whole plant regeneration system, which could be effectively utilized for the production of transgenic finger millet plants.

Effect of seaweed liquid extracts and plant growth regulators on in vitro mass propagation of brinjal (Solanum melongena L.) through hypocotyl and leaf disc explants

The effect of seaweed liquid extracts (SLEs) made from Gracilaria salicornia, Padina gymnospora, Padina boergesenii, Gelidiella acerosa and plant growth regulators (PGRs) were examined on in vitro mass propagation using hypocotyls and leaf disc explants of brinjal (Solanum melongena L.) cultivar Pusa purple long. For the germination bioassay, seeds germinated with 20–40xa0% SLEs exhibited enhanced germination. Initially, hypocotyls and leaf discs were cultured on Murashige and Skoog (MS) medium containing 6-benzylaminopurine, zeatin and thidiazuron for shoot induction. The best responding cytokinin, 6-benzylaminopurine, was employed with different auxins (indole-3-acetic acid, indole-3-butyric acid and 1-naphthaleneacetic acid) for shoot proliferation. In a second experiment, all the four SLEs (10–60xa0%) combined with MS medium were studied for shoot propagation. Augmented shoots transferred to half-strength MS medium and supplemented with auxins and SLEs (10–70xa0%) individually to induce rooting. In these experiments high rate of shoot induction (96.2xa0%), proliferation (6xa0cm) and rooting (95.3xa0%) was found with 20–40xa0% of SLEs. Well-matured plantlets were transferred to soil cups, maintained in a growth chamber for a week to control humidity and then shifted to a greenhouse. This study demonstrated that SLEs could serve as an alternative to phytohormones as they were easy to extract and gave quick and high-frequency mass propagation.

Somatic embryogenesis and regeneration using Gracilaria edulis and Padina boergesenii seaweed liquid extracts and genetic fidelity in finger millet (Eleusine coracana)

The effect of seaweed liquid extracts (SLEs) from Gracilaria edulis and Padina boergesenii was studied on finger millet bioassays. Finger millet seeds were germinated on 0–100xa0% of SLEs. Genotype ‘PR-202’ showed superior response with higher frequency of germination (99.6xa0%), fresh weight (1.1xa0mg seedling−1), shoot length (13.4xa0cm), root number (4.8), and root length (8.6xa0cm) with 60xa0% of G. edulis extract. Influence of SLEs and various phytohormones on somatic embryogenesis and regeneration of finger millet was studied. Shoot apical meristem was inoculated on Murashige and Skoog (MS) medium containing 3–5xa0mgxa0L−1 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4,5-trichlorophenoxyacetic acid individually or in combination of 0.1–1.0xa0mgxa0L−1 kinetin or 10–40xa0% each SLEs of G. edulis or P. boergesenii was used for callus induction and somatic embryogenesis. SLEs of each species were used individually at 20–60xa0% and 25–75xa0% for somatic embryogenesis and regeneration, respectively. The optimum levels of either SLE were found to be 20, 40, and 50xa0% for callus induction, somatic embryogenesis, and regeneration from somatic embryos, respectively. The MS medium containing 4.0xa0mgxa0L−1 2,4-D and 0.5xa0mgxa0L−1 kinetin produced 53.2xa0% somatic embryogenesis in genotype ‘PR-202’ where G. edulis extract improved the somatic embryogenesis to 66.6xa0%. Further, 40xa0% of SLEs emerged somatic embryogenesis 89.8xa0%, regeneration of embryogenic callus 96.3xa0%, and rooting of elongated shoots 97.4xa0%, respectively. Well-grown plantlets were acclimatized in the greenhouse with 94xa0% survival rate. RAPD profiles of in vitro regenerated and mother plants were similar and no somoclonal variation was detected. The current study confirmed that SLEs can be used for somatic embryogenesis and plant regeneration.

Effects of cefotaxime, amino acids and carbon source on somatic embryogenesis and plant regeneration in four Indian genotypes of foxtail millet (Setaria italica L.)

The effects of cefotaxime, amino acids and carbon source on somatic embryogenesis and plant regeneration using mature seeds in four genotypes (‘CO5’, ‘CO7’, ‘TNAU43’ and ‘RS118’) of foxtail millet have been studied. The ‘CO5’ gave a superior response in callus induction, somatic embryogenesis and regeneration. The highest percentage (69.3%) of embryogenic callus induction was obtained in ‘CO5’ on Murashige and Skoog (MS) medium supplemented with 3.5xa0mgxa0L−1 2,4-dichlorophenoxyacetic acid (2,4-D), 1xa0mgxa0L−1 kinetin and 1xa0mgxa0L−1 1-naphthaleneacetic acid (NAA). Somatic embryogenesis and shoot regeneration were influenced by amino acids, carbohydrates and cefotaxime in culture medium. Maximum response of somatic embryo induction and maturation was seen on MS medium containing 3.5xa0mgxa0L−1 2,4-D, 1xa0mgxa0L−1 kinetin and 1xa0mgxa0L−1 NAA, 750xa0mgxa0L−1 proline, 2.0xa0mgxa0L−1 glycine, 150xa0mgxa0L−1 arginine, 800xa0mgxa0L−1 casein enzymatic hydrolyzate, 20xa0gxa0L−1 each sucrose and maltose and 500xa0mgxa0L−1 cefotaxime. The highest frequency of plant regeneration with 21.3 shoots was obtained in ‘CO5’ on MS medium containing 3xa0mgxa0L−1 6-benzylaminopurine, 0.2xa0mgxa0L−1 2,4-D, 750xa0mgxa0L−1 proline, 2.0xa0mgxa0L−1 glycine, 150xa0mgxa0L−1 arginine and 800xa0mgxa0L−1 casein enzymatic hydrolyzate. The highest response of root induction with more roots and longer roots was observed in ‘CO5’ when cultured on half-strength MS medium. The in vitro-regenerated plantlets were carefully transferred to soil cups, maintained in growth chamber for a week, hardened and grown to maturity in the field.

In vitro mass propagation and conservation of Nilgirianthus ciliatus through nodal explants: A globally endangered, high trade medicinal plant of Western Ghats

Abstract Nilgirianthus ciliatus is a globally endangered aromatic slender shrub of Western Ghats with extensive applications in Ayurveda. It is endangered due to its indiscriminate collection and overexploitation to meet the requirements of the pharmaceuticals. This study deals with the preservation of this endangered plant through in vitro nodal culture. Nodal explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) or 6-furfurylaminopurine individually for primary shoot induction. For multiple shoot induction, primary shoots were transferred onto MS medium containing BA individually or in combination with auxins. Clusters of multiple shoots (up to 24.3) occurred with highest frequency (93.2%) on MS medium fortified with BA (3 mg l−1) and indole-3-acetic acid (0.1 mg l−1). In vitro regenerated plantlets were rooted on half-strength MS medium with maximum rooting frequency (82.2%) obtained in the presence of indole-3-butyric acid (1.0 mg l−1). The rooted plantlets were acclimatized to soil with 100% survival rate. Results of this study allowed us to develop an efficient regeneration system that will permit to carry out restoration programmes of N. ciliatus in Western Ghats. In future, this protocol will be an invaluable tool to produce synthetic seeds for cryopreservation and long-term conservation.

Analysis of genetic variation in sorghum (Sorghum bicolor (L.) Moench) genotypes with various agronomical traits using SPAR methods

Genetic variation among 45 genotypes of sorghum (Sorghum bicolor L.) representing seven subpopulations was assessed using three single primer amplification reaction (SPAR) methods viz., inter-simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD) and directed amplification of minisatellite-region DNA (DAMD). Totally 15 ISSR, 8 RAPD and 7 DAMD primers generated 263 amplification products, accounting for 84.6% polymorphism across all the genotypes. The Mantels test of correlation revealed the best correlation between ISSR and cumulative data with a correlation coefficient (r) of 0.84. Assessment of population diversity indicated that the maximum intra population genetic diversity was recorded among high FeZn lines (HFL) having maximum values of Neis genetic diversity (h) (0.244), Shannon information index (I) (0.368) and the percentage of polymorphic loci (Pp) (72.65%) while the corresponding lowest values of 0.074, 0.109 and 17.95% respectively were observed among the members of MDT subpopulation. The mean coefficient of gene differentiation (GST) and the gene flow (Nm) between populations were observed to be 0.396 and 0.7680 respectively. The analysis of molecular variance (AMOVA) suggested that maximum genetic variation exists within populations (95%) than among populations (5%). Thus the information obtained from this study could be utilized in sorghum breeding programmes for the development of varieties with improved nutrition and agronomic values in future.

In vitro propagation and genetic fidelity analysis of alginate-encapsulated Bacopa monnieri shoot tips using Gracilaria salicornia extracts

The scope of the present study is to investigate the effect of plant growth regulators and seaweed liquid extracts (SLEs) on in vitro regeneration of the Ayurvedically important memory plus plant Bacopa monnieri using shoot tips. Sodium alginate-encapsulated shoot tips were cultured on Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP), kinetin, zeatin, 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA), individually for shoot initiation. The effect of Gracilaria salicornia and Kappaphycus alvarezii SLEs (20–80xa0%) for shoot induction and regeneration using shoot tips was also studied. MS medium containing 1.0xa0mgxa0L−1 BAP and 0.5xa0mgxa0L−1 NAA showed highest multiple shoot induction frequency 95.8xa0%, mean number 147.8, and mean length 12.5xa0cm of shoots where G. salicornia extract at 60xa0% showed 85.9xa0% multiple shoot induction with 145.6 shoots and mean length 12.3xa0cm after 5xa0weeks of incubation. An effective rooting frequency 84.1xa0% was observed on half-strength MS medium containing 0.1xa0mgxa0L−1 IAA and where 25xa0% of G. salicornia extract showed 82.2xa0% of rooting after 1xa0week of incubation. In vitro-rooted plants were acclimatized in soil cups and maintained in greenhouse with natural conditions. In vitro-propagated and mother plants were analyzed using random amplified polymorphic DNA markers reveled that no genetic variation between control plants and in vitro-regenerated plants using SLEs. These results showed that G. salicornia extracts can be used as PGRs for in vitro propagation of B. monnieri and it is a simple, rapid mass propagation method.

Analysis of propagation of Bacopa monnieri (L.) from hairy roots, elicitation and Bacoside A contents of Ri transformed plants

Agrobacterium rhizogenes mediated transformation has been experimented in leaf explants of the memory herb Bacopa monnieri in order to assess the regeneration potential of hairy roots (HR) followed by the elicitation of transformed plants for increased Bacoside A production. Out of the four strains tested, A4 and MTCC 532 derived HR exhibited regrowth in MS basal medium while MTCC 2364 derived HR showed regeneration in MS medium supplemented with suitable phyto hormones. R1000 derived HR possessed no regeneration potential. Comparable to A4, MTCC 532 derived HR displayed maximum regrowth frequency of about 85.71xa0±xa01.84xa0% with an increase in biomass to threefold. Therefore, five HR plant lines (MTCC 532 derived) were generated and maintained in MS basal liquid medium in which HR3 topped the others in producing a huge biomass of about 67.09xa0±xa00.66xa0g FW. PCR amplification and southern hybridization analysis of rol A gene (280xa0bp) has been performed in order to confirm the transformation process. Moreover, HR3 plant line has accumulated highest total phenolic content of about 165.68xa0±xa00.82xa0mg GAE/g DW and highest total flavonoid content of about 497.78xa0±xa00.57xa0mg QRE/g DW when compared to other lines and untransformed controls. In addition, HR3 plant extract showed 85.58xa0±xa00.14xa0% of DPPH (2, 2-diphenyl-1-picryl hydrazyl) inhibition displaying its reliable anti oxidant potential. Further on elicitation with 10xa0mg/L chitosan for 2xa0weeks, HR3 has produced 5.83xa0% of Bacoside A which is fivefold and threefold increased production when compared to untransformed and transformed unelicited controls respectively. This is the first report on eliciting HR plants for increased metabolite accumulation in B. monnieri.

Analysis of population structure and genetic diversity in an exotic germplasm collection of Eleusine coracana (L.) Gaertn. using genic-SSR markers

Finger millet (Eleusine coracana (L.) Geartn.) is one of the important small millets serves as a food security crop because of its high nutritional values. The complex tetraploid genome of finger millet requires a large number of informative, functional DNA markers for different applications in genetics and breeding. Yet, less number of simple sequence repeat (SSR) markers have been developed from expressed sequence tags in finger millet. In the present study, 56 new genic SSR markers were developed from publicly available drought related ESTs. The 43 polymorphic markers were used to evaluate polymorphism, revealed a range of PIC value 0.41 to 0.79. Our results suggest that, analyzed genotypes have high genetic diversity with an average gene diversity (h) of 0.176 and Shannons information index (I) of 0.315. We conclude that there was a higher gene exchange within populations, by the value of overall gene flow (Nm) of 0.7721. The unweighted pair group method with arithmetic mean and neighbor joining dendrogram generated three main clusters to differentiate genotypes and these results were also confirmed by PCA and PCoA analysis. The high genetic diversity (77%) was found within the populations in the analysis of molecular variance. A Bayesian model-based cluster analysis evidenced a high extent of admixture between the gene pools from the different geographical origins. Population based cluster analyses pointed out a strong pattern of isolation by distance. Overall, these results underscored that this study showed a significantly high level of polymorphism, adequate genetic diversity and population structure which expand the modern genetic resources and its utility in various applications in genetics and genomics including association mapping and breeding.